1. Are you sure you have the correct flasks which are cell biocompatible for adherent cells?

2. Did you thaw them quickly, as soon as aliquot is liquid, transfer directly to warmed medium?

3. It is a transformed cell line with Simian Virus, thus have you grown them at the correct temperature?

4. 13000rpm is far too high for any cell type, 1000rpm for 1-2 minutes is sufficient for spinning down cells to a pellet.

1. Are you sure you have the correct flasks which are cell biocompatible for adherent cells?

2. Did you thaw them quickly, as soon as aliquot is liquid, transfer directly to warmed medium?

3. It is a transformed cell line with Simian Virus, thus have you grown them at the correct temperature?

4. 13000rpm is far too high for any cell type, 1000rpm for 1-2 minutes is sufficient for spinning down cells to a pellet.

Apart from the points listed above (especially the use of TC treated flasks for adherent cells) one reason that the cells are not adhering after defrosting is that they are already dead.

The reasons for this might be:

a) Cells were stored for a prolonged time at -20/-80 degrees C. before transferring to liquid nitrogen.

b) Cells were frozen down too rapidly or cryo-protectant (such as 10% DMSO) was not added.

I am a little puzzled by the line "I thaw them in High Glucose medium with 10% FBS and 0.1% penicillin". I assume that the cells were defrosted in the cryo-vials they were stored in liquid nitrogen in and that after the cells were defrosted they were transferred to the above media?

You mention that the cells were from ATCC. Are you having problems with cells that  were shipped to you from ATCC or have they been expanded/frozen in your lab and it is these cells that fail to adhere? If it is the former you should contact ATCC (with the lot. number of the cells) and ask their advice (or if any problems have been reported by others with this cell line). If it is the latter you should see if you have any different passage levels of these cells (perhaps a Master bank vial?) and see if these too fail to adhere.

Sorry I can't be more help.

Gary

Thanks a lot for your detailed replies Mr. Vehid Salih and Gary.

I received cells from ATCC in morning and i at once kept them in liquid nitrogen for whole day then I thaw in water bath 37'C for less then 2mins and aliquot in 9ml High Glucose medium(with FBS and penicilin). Then I centrifuge them at rpm 13000 for 3mins . Then after changing the media I transferred them into TC treated non pyrogenic  flask and incubated at 37'C with 5% CO2. 

Our lab order cells through distributors. And it took almost 2 months to recieve cells. May be they were stored for a prolonged time at -20/-80 degrees C.

I have already contacted the atcc people, but they didn't reply yet.

I am still trying by passing them to new flasks after centrifuge. 

Sir Vehid, I will try 1000rpm for sure. Thanks for advice.

Thanks,

I suspect (as Vehid has mentioned) that the 13000 rpm spin did them no good. The "g" force will be dependent on the rotor size  but is likely to have exceeded 10 000 g ( I spin at 1500 rpm for 5 min in an Eppendorf bench top centrifuge and even this is a little high, you are spinning at ~ 400x the gravity of Jupiter).

People do tend to centrifuge the cells after trypsinisation but to be honest I count the cells in the trypsin and add the desired amount directly to the media in the new flasks. Trypsin is neutralised pretty quickly by the FBS in the media so centrifugation is not necessary (this is the reason cells are rinsed in PBS prior to trypsinisation  - to remove trace FBS from the cells). However please do stick to local protocols.

  I would have thought some of the cells would still have survived this (do you still have the original flask? if so check for any adherent cells, they may be few and far between). Unfortunately if the cells are dead then passaging them into different flasks won't be much help :(

Anyway, good luck.

Gary

Sounds like they are dead unfortunately. Like others, I suggests a much gentler spin (for HEKs, I usually do 150g for 2 min), or even not centrifuging at all, i.e. simply suspending the thawed cells in prewarmed media, letting them adhere overnight, and then fully changing the media the next day to remove the cryoprotectant. 

Thanks alot for all the help.. I really appreciate it.. ATCC is sending new cells again. And I will keep in mind all the points we discussed.. 

I do not believe that 13.000 rmp for 5 min are a strong spin forfor SVEC4-10 cells. We used 20.000 rpm during 6 min and got very good ECs. The point could be related to the osmolarity of the nutrient solution or the short time maintaining cells in liquid nitrogen. Any answer could be only speculation. Try to ask to the provider for their treatment.

Yes,I guess you have a valid point  Mr. Marco.. As i just got email from providers and they mentioned same point... The media I used for thawing and passage was with 3.5gm/L sodium bicarbonate and then I incubated cells at 5% CO2. But I was suppose to use Media with 1.5gm/L of sodium bicarbonate for 5%CO2.

Although one can use meida with 3.5gm/L of sodium bicarbonate but then cells should be placed in 10% CO2. 

Thanks alot for you opinion and time.. :)